PROJECT SUMMARY Cancer affects 39.6% of Americans at some point during their lifetime. Solid tumor microenvironments are characterized by a disorganized, leaky vasculature that promotes regions of low oxygenation. In fact, tumor hypoxia is a key predictor of poor treatment outcome for all radiotherapy, chemotherapy and surgery procedures, as well as a hallmark of metastatic potential. In particular, tumor cell resistance to radiotherapy is 3 fold increased in anoxic cells and even very small tumors comprise 10-30% of hypoxic regions in the form of chronic and/or transient hypoxia fluctuating over course of seconds to days. Recently, lipid-stabilized oxygen microbubbles (OMBs) have been used in vivo to relieve tumor hypoxia in sonodynamic therapy when injected directly in the tumors, as well as shown to sustain asphyxiated animals for over two hours when injected intra-peritoneally. Our preliminary data supports our hypothesis that oxygen microbubbles could also be used to relieve tumor hypoxia during radiotherapy and significantly improve treatment outcome. In addition, there has been no systemic OMB delivery demonstration to date for tumor hypoxia modulation with OMBs, due in part to the difficulty of measuring hypoxia in vivo reliably in combination with these administrations. We hypothesize that we can guide specially formulated OMBs via ultrasound imaging, and preferentially release oxygen, in the tumor for radiosensitization to significantly improve the radiotherapy therapeutic ratio. In order to test our hypothesis, will optimize microbubble formulations and administration parameters, evaluate biological mechanisms and kinetics, and validate our hypothesis both in a rodent model and then in a translational large animal model across two of the leading veterinary schools in the country. To achieve these goals, we approach this project with a collaborative team of leading experts in the fields of microbubbles, oxygen transport, radiation oncology, and tumor biology.